Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117+/-12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9+/-0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106+/-11 nmol HCHO/min/mg SLO, and 3.2+/-0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.     

N-dealkylation of aminopyrine catalyzed by soybean lipoxygenase in the presence of hydrogen peroxide

A hypothesis that lipoxygenase may mediate N-dealkylation of xenobiotics was investigated using the prototype drug aminopyrine and soybean lipoxygenase as a model enzyme in the presence of hydrogen peroxide. Formaldehyde production as a result of N-demethylation of aminopyrine exhibited pH optimum of 6.5. The reaction was dependent on the incubation time, amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. Under the experimental conditions employed, the specific activity for N-demethylation of aminopyrine was found to be 823 ± 93 nmoles per min/mg protein or 89 ± 10 nmoles per min/nmole of enzyme. The reaction was significantly inhibited by nordihydroguaiaretic acid and gossypol, the classical inhibitors of lipoxygenase. Spectrophotometric analyses indicated the generation of a nitrogen-centered free-radical cation as the initial oxidation product of aminopyrine. The rate of accumulation of this radical species was also dependent on pH, the amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. The radical production was markedly suppressed by ascorbate, glutathione, and dithiothreitol in a concentration-dependent manner. Preliminary data gathered for the oxidation of other chemicals indicated that the lipoxygenase exhibits a unique substrate specificity. Collectively, the evidence presented suggests for the first time that lipoxygenase pathway may be involved in N-demethylation of aminopyrine and other chemicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 175–183, 1998     

Lipoxygenase-mediated hydrogen peroxide-dependent N-demethylation of N,N-dimethylaniline and related compounds

To date, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases. In this study we investigated the ability of non-heme iron proteins, namely soybean lipoxygenase (SLO) and human term placental lipoxygenase (HTPLO) to mediate N-demethylation of N,N-dimethylaniline (DMA) and related compounds in the presence of hydrogen peroxide. In addition to being hydrogen peroxide dependent, the reaction was also dependent on incubation time, concentration of enzyme and DMA and the pH of the medium. Using Nash reagent to estimate formaldehyde production, we determined the specific activity for SLO mediated N-demethylation of DMA to be 200 + 18 nmol HCHO/min per mg protein or 23 +/- 2 nmol/min per nmol of enzyme, while that of HTPLO was 33 +/- 4 nmol HCHO/min per mg protein. Nordihydroguaiaretic acid (NDGA), a classical inhibitor of lipoxygenase (LO), as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of production of formaldehyde from DMA by SLO. Besides N,N-dimethylaniline, N-methylaniline, N,N,N',N'-tetramethylbenzidine, N,N-dimethyl-p-phenylenediamine, N,N-dimethyl-3-nitroaniline and N,N-dimethyl-p-toluidine were also demethylated by SLO. The formation of a DMA N-oxide was not detected. Preliminary experiments suggested SLO-mediated hydrogen peroxide-dependent S-dealkylation of methiocarb or O-dealkylation of 4-nitroanisole does not occur.     

Hydroperoxidase activity of lipoxygenase: hydrogen peroxide-dependent oxidation of xenobiotics

Since H2O2 is one of the major biologically available peroxides, its ability to support hydroperoxidase activity of highly purified soybean lipoxygenase was examined by monitoring co-oxidation of selected xenobiotics. All of the eight chemicals tested were found to be oxidized in the presence of H2O2. Tetramethylbenzidine oxidation was completely inhibited by the classical lipoxygenase inhibitor nordihydroguaiaretic acid. The reaction was enzymatic in nature and exhibited a acidic pH optimum. The data clearly indicate, for the first time, that H2O2 can efficiently replace fatty acid hydroperoxide in a xenobiotic oxidation reaction medicated by the hydroperoxidase activity of lipoxygenase.     

Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117±12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9±0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106±11 nmol HCHO/min/mg SLO, and 3.2±0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.     

Formation of homovanillic acid dimer by enzymatic or Fenton system - catalyzed oxidation

Homovanillic acid is the most extensively employed reagent for the fluorometric detection of peroxidase. However, the assays based on the determination of the oxidation product of homovanillic acid do not allow a selective detection of the enzyme, because chemical or physical factors can interfere with the fluorometric determination. The aim of this work was to verify if other enzymatic or non-enzymatic systems might catalyze the homovanillic acid oxidation. The reaction was investigated by spectrophotometric and fluorometric assays; HPLC analysis was used to separate homovanillic acid from its oxidation product and to obtain information on the oxidation process. The results obtained showed that soybean lipoxygenase in the presence of hydrogen peroxide can oxidize homovanillic acid with the formation, by an o,o'-biphenyl linkage, of the corresponding dimer as the sole reaction product. The reaction followed Michaelis-Menten kinetics, for both homovanillic acid and hydrogen peroxide. Other systems, such as cytochrome c/H(2)O(2) and Fenton reagents, were also able to oxidize homovanillic acid to its dimer. It can be affirmed that possible interference by other oxidative systems - that could be present in the biological materials tested - should be considered in assays of peroxidase activity based on the detection of the dimer of homovanillic acid.     

Hydrogen peroxide: a potent activator of dioxygenase activity of soybean lipoxygenase

Hydrogen peroxide, an ubiquitous biologically occurring peroxide, was found to stimulate the dioxygenase activity of soybean lipoxygenase at the physiologically attainable concentration. The increase in enzyme specific activity was directly proportional to hydrogen peroxide concentration up to 0.5 nM. A decrease in the stimulation of dioxygenase activity was observed at higher concentrations. At low enzyme concentration up to 28-fold stimulation was noted when the formation of lipid hydroperoxide was monitored spectrophotometrically. The stimulation was further confirmed by increased oxygen uptake. It is proposed that the mechanism for in vivo activation involves hydrogen peroxide.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins.

The oxidation of xenobiotics by the hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins was studied. These produced an inhibitory effect on xenobiotics oxidation, based on their degree of hydrophobicity and the charge (isoproterenol < 4-methyl-catechol (4MC) < 4-tert-butylcatechol (TBC) < 4-tert-octylcatechol (TOC)). This inhibitory effect was due to the complexation of xenobiotics in the hydrophobic cavity of cyclodextrins. The complexation constant Kc was calculated by nonlinear regression of the inhibition curves obtained in the presence of cyclodextrins, and the values obtained were 400, 16,250, and 35,127 M-1 for 4MC, TBC, and TOC, respectively. The validity of these values was checked at different points of the Michaelis-Menten saturation curve, and a sigmoidal inhibition curve was obtained at the saturating concentration of the o-diphenol, TBC, with no change in the Kc value. This demonstrates the validity of the equations used to calculate Kc for the complete range of the Michaelis-Menten equation.     

Lipoxygenases of animals: changes in lipoxygenase activity from reticulocytes during interaction with blood plasma lipoproteins

A homogeneous sample of lipoxygenase from rabbit reticulocyte cytosol was obtained. The rate of exogenous arachidonate oxidation by reticulocyte lipoxygenase was found to increase in the presence of various blood plasma lipoproteins of mammals. The rate of arachidonate oxidation by soybean lipoxygenase in the presence of these lipoproteins was either unaffected or decreased. The lipoproteins immobilized the substrate; however, preliminary "saturation" of the lipoproteins by a manyfold excess of non-oxidizable oleate did not eliminate the enzyme activation by the lipoproteins. It was concluded that lipoxygenase activation is due to conformational changes of the enzyme during its interaction with membrane components.     

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.     

Hydroperoxidase I catalyzes peroxidative activation of 3,3'-dichlorobenzidine to a mutagen in Salmonella typhimurium

Dichlorobenzidine can be peroxidatively activated in Salmonella typhimurium Ames tester strains. Mutagenicity is observed when an S. typhimurium strain which is sensitive to frame-shift mutagens is incubated with dichlorobenzidine and hydrogen peroxide. In this paper, we show that the bacterial enzyme, hydroperoxidase I, is responsible for much of this activation. We constructed isogenic tester strains which lack hydroperoxidase I or II, due to Tn10 insertions in the structural genes encoding these proteins. Hydrogen peroxide-dependent mutagenicity of dichlorobenzidine was measured in each strain. A tester strain lacking hydroperoxidase I activity was much less sensitive than was the parent strain. When hydroperoxidase I activity was restored in this strain, via a plasmid-borne copy of the gene encoding the Escherichia coli protein, sensitivity to peroxide-dependent dichlorobenzidine mutagenicity was enhanced.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Cross-linking of horseradish peroxidase adsorbed on polycationic films: utilization for direct dye degradation

Horseradish peroxidase (HRP) was immobilized on the polyaniline (PANI) grafted polyacrylonitrile (PAN) films. The maximum HRP immobilization capacity of the PAN-g-PANI-3 film was 221?μg/cm(2). The HRP-immobilized PAN-g-PANI-3 film retained 79?% of the activity of the same quantity free enzyme. The HRP-immobilized PAN-g-PANI-3 film was operated for the decolorization of two different benzidine-based dyes in the presence of hydrogen peroxide. The maximum decolorization grade was obtained at pH 6.0 for both dyes. The HRP-immobilized PAN-g-PANI-3 film was very effective for removal of Direct Blue-53 compared to Direct Black-38 from aqueous solutions. The immobilized HRP exhibited high resistance to proteolysis by trypsin compared to the free counterpart. Immobilized HRP preserved 83?% of its original activity even after 8?weeks of storage at 4?°C, while the free enzyme lost its initial activity after 3?weeks of storage period.     

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.     

A kinetic model for lipoxygenases based on experimental data with the lipoxygenase of reticulocytes

A comprehensive kinetic model for lipoxygenase catalysis is proposed which includes the simultaneous occurrence of dioxygenase and hydroperoxidase activities and is based on the assumption of a single binding site for substrate fatty acid and product. The aerobic reaction of purified lipoxygenase from rabbit reticulocytes with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate was studied. The rate constants and the dissociation constants of this enzyme were calculated for the model from progress curves; the model describes correctly the experimental data. The following kinetic features of the reticulocyte enzyme are assumed to apply generally to lipoxygenases. (a) The enzyme shows autoactivation by its product. (b) The rate-limiting step is the hydrogen abstraction. (c) Both substrate fatty acid and its product are competitive inhibitors of the lipoxygenase. (d) Lowering the oxygen concentration enhances the degree of substrate inhibition, whereas product inhibition is not influenced. (e) If substrate is in excess the oxygen concentration determines the share of dioxygenase and hydroperoxidase activities of the enzyme. As predicted from the model it was found that at low concentrations of oxygen the regio- and stereo-specificities of the dioxygenation are diminished. During the autoactivation phase the steady-state approximation does not hold.     

Isolation and some properties of dioxygenase and co-oxidase activities of adult human liver cytosolic lipoxygenase

The evidence presented here constitutes the first report on the occurrence of lipoxygenase (LO) activity in the adult human liver. LO activity was isolated free of hemoglobin from the whole liver cytosol by affinity chromatography using a concanavalin-A sepharose 4B column, and some properties of its dioxygenase and co-oxidase activities were examined. High-pressure liquid chromatography (HPLC) analyses of arachidonic acid metabolites suggested the presence of 5-, 12-, and 15-LO activities in the human liver. Linoleic acid was converted into 13-hydroperoxyoctadecadienoic acid. The dioxygenase activity with a V_max value of 1.74 μmoles/min/mg protein and a K_m value of 0.48 mM was noted in the presence of different concentrations of linoleic acid at pH 10. The activity was markedly stimulated by the presence of calcium, ATP, hydrogen peroxide, and KCl in the assay medium. Under optimum conditions, all the xenobiotics tested were co-oxidized by the enzyme preparations in the presence of linoleic acid. Kinetic data obtained for benzidine oxidation yielded a K_m value of 0.53 mM and a V_max value of 90.9 nmoles/min/mg protein. At present, the significance of these findings in in vivo toxicity of benzidine is unknown. The linoleic acid-dependent dioxygenase and co-oxidase activities were thermolabile and inhibited by micromolar concentrations of several classical LO inhibitors, further confirming the involvement of LO in these reactions. © 1997 John Wiley & Sons, Inc.